Byung Cheol Ahn

Use of pre-s protein-containing hepatitis B virus surface antigens and a powerful adjuvant to develop an immune therapy for chronic hepatitis B virus infection

ABSTRACT A hepatitis B virus (HBV) vaccine has been developed using a new adjuvant and HBV surface antigens produced from a CHO cell line. The purified HBV surface antigens are composed of L protein, M protein, and S protein in a mixture of 20- and 40-nm-diameter particles and filamentous forms. This HBV surface antigen, formulated with L-pampo, a proprietary adjuvant, induced 10 times more antibody than the same antigen with alum and was capable of inducing strong immune responses in three different HBV transgenic mice. In spite of the presence of a large amount of HBV antigen in the blood, no antibody against HBV surface antigen was normally detected in these transgenic mice. After immunization, the HBV antigen was also cleared from the blood.

Combination of TLR1/2 and TLR3 ligands enhances CD4 + T cell longevity and antibody responses by modulating type I IFN production

Despite the possibility of combining Toll-like receptor (TLR) ligands as adjuvants to improve vaccine efficacy, it remains unclear which combinations of TLR ligands are effective or what their underlying mechanisms may be. Here, we investigated the mechanism of action of L-pampo, a proprietary adjuvant composed of TLR1/2 and TLR3 ligands. L-pampo dramatically increased humoral immune responses against the tested target antigens, which was correlated with an increase in follicular helper T cells and the maintenance of antigen-specific CD4+ T cells. During the initial priming phase, in contrast to the induction of type I interferon (IFN) and pro-inflammatory cytokines stimulated by polyI:C, L-pampo showed a greatly diminished induction of type I IFN, but not of other cytokines, and remarkably attenuated IRF3 signaling, which appeared to be critical to L-pampo-mediated adjuvanticity. Collectively, our results demonstrate that the adjuvant L-pampo contributes to the promotion of antigen-specific antibodies and CD4+ T cell responses via a fine regulation of the TLR1/2 and TLR3 signaling pathways, which may be helpful in the design of improved vaccines.

Overproduction of recombinant human mannose-binding lectin (MBL) in Chinese hamster ovary cells

Mannose-binding lectin (MBL) is an important serum protein that functions in the innate immune system and has been considered to have therapeutic potential in MBL replacement therapies for patients with deficient or low levels of MBL. In this study, we established a Chinese hamster ovary (CHO) cell line that overexpresses the recombinant human MBL (rhMBL) protein. In an 11-day batch culture process using a 30-L bioreactor (20-L working volume) and serum-free medium, these cells could produce over 226 mg/L of rhMBL protein. The recombinant protein was then purified to homogeneity from the culture supernatant using a three-step chromatographic procedure that resulted in a recovery rate of approximately 55%. This purified rhMBL protein adopted oligomeric bouquet-like structures that were similar to those of native MBL present in human blood, and these oligomeric structures were reported to be critical in MBL functions. We further demonstrated in carbohydrate binding and complementation activation assays that this rhMBL protein was functionally active with very similar dissociation constants and half maximal effective concentrations to those of native MBL.

Abstract B25: Visualization of immune response to Hepatitis B vaccination by in vivo mouse imaging

By taking advantage of bioluminescence imaging to monitor luciferase expressing splenocytes from the luciferase transgenic mouse, we visulaized the enhancement of immune response against hepatitis B virus (HBV) vaccine with adjuvants. To visualize antigen in vivo, large hepatitis B virus antigen (L-HBsAg) was labeled with radioiodine (125I). B6 mice were intramuscularly vaccinated. The localization of 125I-L-HBsAg was monitored for 5 weeks using animal SPECT/CT. To monitor the immune response, the luciferase expressing splenocytes were injected intravenously into immunized B6 mice. Bioluminescence signals from splenocytes were measured by IVIS 100 system. Localization of inoculated L-HBsAg was successfully monitored using animal SPECT/CT. L-HBsAg was lasted for 5 weeks and diminished. In addition, the injected splenocytes were successfully visualized in immunized mice, homing to spleen within 30 minutes and were accumulated in lymph nodes within 5 hours. Accumulation of splenocytes at vaccination site was observed within 24 hours. Moreover, after 10 hours, mouse vaccinated with antigen and two adjuvants showed 4 times more accumulation of splenocytes at vaccination site compared to mouse vaccinated with antigen only. Six days later, mouse vaccinated with antigen and two adjuvants showed 1.7-4.73 fold increased luciferase intensity of splenocytes at spleen, lymph nodes and vaccination site compared to mouse vaccinated with antigen only. In conclusion, in vivo real-time bioluminescent monitoring of splenocytes homing and proliferation against vaccination successfully provides efficiency of adjuvants. Our imaging system can be used for evaluation of efficacy of vaccination by enhanced the proliferation and activation of splenocytes near the vaccination site. Note: This abstract was not presented at the conference. Citation Format: Ha Kim, Keejong Hong, Byung Cheol Ahn, Jung Sun Yum, Hyewon Youn. Visualization of immune response to Hepatitis B vaccination by in vivo mouse imaging. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B25.